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1. Synergistic co‐utilization of biomass‐derived sugars enhances aromatic amino acid production by engineered Escherichia coli

   https://doi.org/10.1002/bit.28585  

   Liu, Arren ; Machas, Michael ; Mhatre, Apurv ; Hajinajaf, Nima ; Sarnaik, Aditya ; Nichols, Nancy ; Frazer, Sarah ; Wang, Xuan ; Varman, Arul M. ; Nielsen, David R. ( November 2023 , Biotechnology and Bioengineering)

   Efficient co‐utilization of mixed sugar feedstocks remains a biomanufacturing challenge, thus motivating ongoing efforts to engineer microbes for improved conversion of glucose−xylose mixtures. This study focuses on enhancing phenylalanine production by engineeringEscherichia colito efficiently co‐utilize glucose and xylose. Flux balance analysis identified E4P flux as a bottleneck which could be alleviated by increasing the xylose‐to‐glucose flux ratio. A mutant copy of the xylose‐specific activator (XylR) was then introduced into the phenylalanine‐overproducingE. coliNST74, which relieved carbon catabolite repression and enabled efficient glucose−xylose co‐utilization. Carbon contribution analysis through¹³C‐fingerprinting showed a higher preference for xylose in the engineered strain (NST74X), suggesting superior catabolism of xylose relative to glucose. As a result, NST74X produced 1.76 g/L phenylalanine from a model glucose−xylose mixture; a threefold increase over NST74. Then, using biomass‐derived sugars, NST74X produced 1.2 g/L phenylalanine, representing a 1.9‐fold increase over NST74. Notably, and consistent with the carbon contribution analysis, thexylR*mutation resulted in a fourfold greater maximum rate of xylose consumption without significantly impeding the maximum rate of total sugar consumption (0.87 vs. 0.70 g/L‐h). This study presents a novel strategy for enhancing phenylalanine production through the co‐utilization of glucose and xylose in aerobicE. colicultures, and highlights the potential synergistic benefits associated with using substrate mixtures over single substrates when targeting specific products.

    

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2. The Arabidopsis xylosyltransferases, XXT3 , XXT4 , and XXT5, are essential to complete the fully xylosylated glucan backbone XXXG ‐type structure of xyloglucans

   https://doi.org/10.1111/nph.18851  

   Zhang, Ning ; Julian, Jordan D. ; Yap, Cheng Ern ; Swaminathan, Sivakumar ; Zabotina, Olga A. ( April 2023 , New Phytologist)

   Although most xyloglucans (XyGs) biosynthesis enzymes have been identified, the molecular mechanism that defines XyG branching patterns is unclear. Four out of five XyG xylosyltransferases (XXT1, XXT2, XXT4, and XXT5) are known to add the xylosyl residue from UDP‐xylose onto a glucan backbone chain; however, the function of XXT3 has yet to be demonstrated.

   Singlexxt3and triplexxt3xxt4xxt5mutantArabidopsis(Arabidopsis thaliana) plants were generated using CRISPR‐Cas9 technology to determine the specific function of XXT3.

   Combined biochemical, bioinformatic, and morphological data conclusively established for the first time that XXT3, together with XXT4 and XXT5, adds xylosyl residue specifically at the third glucose in the glucan chain to synthesize XXXG‐type XyGs. We propose that the specificity of XXT3, XXT4, and XXT5 is directed toward the prior synthesis of the acceptor substrate by the other two enzymes, XXT1 and XXT2. We also conclude that XXT5 plays a dominant role in the synthesis of XXXG‐type XyGs, while XXT3 and XXT4 complementarily contribute their activities in a tissue‐specific manner.

   The newly generatedxxt3xxt4xxt5mutant produces only XXGG‐type XyGs, which further helps to understand the impact of structurally deficient polysaccharides on plant cell wall organization, growth, and development.

    

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3. Hyaluronan synthases; mechanisms, myths, & mysteries of three types of unique bifunctional glycosyltransferases

   https://doi.org/10.1093/glycob/cwad075  

   DeAngelis, Paul L. ; Zimmer, Jochen ( September 2023 , Glycobiology)

   Hyaluronan (HA), the essential [-3-GlcNAc-1-β-4-GlcA-1-β-]n matrix polysaccharide in vertebrates and molecular camouflage coating in select pathogens, is polymerized by “HA synthase” (HAS) enzymes. The first HAS identified three decades ago opened the window for new insights and biotechnological tools. This review discusses current understanding of HA biosynthesis, its biotechnological utility, and addresses some misconceptions in the literature.

   HASs are fascinating enzymes that polymerize two different UDP-activated sugars via different glycosidic linkages. Therefore, these catalysts were the first examples to break the “one enzyme/one sugar transferred” dogma. Three distinct types of these bifunctional glycosyltransferases (GTs) with disparate architectures and reaction modes are known. Based on biochemical and structural work, we present an updated classification system. Class I membrane-integrated HASs employ a processive chain elongation mechanism and secrete HA across the plasma membrane. This complex operation is accomplished by functionally integrating a cytosolic catalytic domain with a channel-forming transmembrane region. Class I enzymes, containing a single GT family-2 (GT-2) module that adds both monosaccharide units to the nascent chain, are further subdivided into two groups that construct the polymer with opposite molecular directionalities: Class I-R and I-NR elongate the HA polysaccharide at either the reducing or the non-reducing end, respectively. In contrast, Class II HASs are membrane-associated peripheral synthases with a non-processive, non-reducing end elongation mechanism using two independent GT-2 modules (one for each type of monosaccharide) and require a separate secretion system for HA export. We discuss recent mechanistic insights into HA biosynthesis that promise biotechnological benefits and exciting engineering approaches.

    

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4. Synthesis of polyketides from low cost substrates by the thermotolerant yeast Kluyveromyces marxianus

   https://doi.org/10.1002/bit.26976  

   McTaggart, Tami L. ; Bever, Danielle ; Bassett, Shane ; Da Silva, Nancy A. ( April 2019 , Biotechnology and Bioengineering)

   Kluyveromyces marxianusis a promising nonconventional yeast for biobased chemical production due to its rapid growth rate, high TCA cycle flux, and tolerance to low pH and high temperature. UnlikeSaccharomyces cerevisiae, K. marxianusgrows on low‐cost substrates to cell densities that equal or surpass densities in glucose, which can be beneficial for utilization of lignocellulosic biomass (xylose), biofuel production waste (glycerol), and whey (lactose). We have evaluatedK. marxianusfor the synthesis of polyketides, using triacetic acid lactone (TAL) as the product. The 2‐pyrone synthase (2‐PS) was expressed on a CEN/ARS plasmid in three different strains, and the effects of temperature, carbon source, and cultivation strategy on TAL levels were determined. The highest titer was obtained in defined 1% xylose medium at 37°C, with substantial titers at 41 and 43°C. The introduction of a high‐stability 2‐PS mutant and a promoter substitution increased titer four‐fold. 2‐PS expression from a multi‐copy pKD1‐based plasmid improved TAL titers a further five‐fold. Combining the best plasmid, promoter, and strain resulted in a TAL titer of 1.24 g/L and a yield of 0.0295 mol TAL/mol carbon for this otherwise unengineered strain in 3 ml tube culture. This is an excellent titer and yield (on xylose) before metabolic engineering or fed‐batch culture relative to other hosts (on glucose), and demonstrates the promise of this rapidly growing and thermotolerant yeast species for polyketide production.

    

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5. Directed evolution of Zymomonas mobilis sugar facilitator Glf to overcome glucose inhibition

   https://doi.org/10.1093/jimb/kuab066  

   Kurgan, Gavin ; Onyeabor, Moses ; Holland, Steven C. ; Taylor, Eric ; Schneider, Aidan ; Kurgan, Logan ; Billings, Tommy ; Wang, Xuan ( September 2021 , Journal of Industrial Microbiology and Biotechnology)

   Cellular import of D-xylose, the second most abundant sugar in typical lignocellulosic biomass, has been evidenced to be an energy-depriving process in bacterial biocatalysts. The sugar facilitator of Zymomonas mobilis, Glf, is capable of importing xylose at high rates without extra energy input, but is inhibited by D-glucose (the primary biomass sugar), potentially limiting the utility of this transporter for fermentation of sugar mixtures derived from lignocellulose. In this work we developed an Escherichia coli platform strain deficient in glucose and xylose transport to facilitate directed evolution of Glf to overcome glucose inhibition. Using this platform, we isolated nine Glf variants created by both random and site-saturation mutagenesis with increased xylose utilization rates ranging from 4.8-fold to 13-fold relative to wild-type Glf when fermenting 100 g l–1 glucose–xylose mixtures. Diverse point mutations such as A165M and L445I were discovered leading to released glucose inhibition. Most of these mutations likely alter sugar coordinating pocket for the 6-hydroxymethyl group of D-glucose. These discovered glucose-resistant Glf variants can be potentially used as energy-conservative alternatives to the native sugar transport systems of bacterial biocatalysts for fermentation of lignocellulose-derived sugars.

    

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