OptSSeq explores enzyme expression and function landscapes to maximize isobutanol production rate.

Efficient microbial production of the next-generation biofuel isobutanol (IBA) is limited by metabolic bottlenecks. Overcoming these bottlenecks will be aided by knowing the optimal ratio of enzymes for efficient flux through the IBA biosynthetic pathway. OptSSeq (Optimization by Selection and Sequencing) accomplishes this goal by tracking growth rate-linked selection of optimal expression elements from a combinatorial library. The 5-step pathway to IBA consists of Acetolactate synthase (AlsS), Keto-acid reductoisomerase (KARI), Di-hydroxy acid dehydratase (DHAD), Ketoisovalerate decarboxylase (Kivd) and Alcohol dehydrogenase (Adh). Using OptSSeq, we identified gene expression elements leading to optimal enzyme levels that enabled theoretically maximal productivities per cell biomass in Escherichia coli. We identified KARI as the rate-limiting step, requiring the highest levels of enzymes expression, followed by AlsS and AdhA. DHAD and Kivd required relatively lower levels of expression for optimal IBA production. OptSSeq also enabled the identification of an Adh enzyme variant capable of an improved rate of IBA production. Using models that predict impacts of enzyme synthesis costs on cellular growth rates, we found that optimum levels of pathway enzymes led to maximal IBA production, and that additional limitations lie in the E. coli metabolic network. Our optimized constructs enabled the production of ~3 g IBA per hour per gram dry cell weight and was achieved with 20 % of the total cell protein devoted to IBA-pathway enzymes in the molar ratio 2.5:6.7:2:1:5.2 (AlsS:IlvC:IlvD:Kivd:AdhA). These enzyme levels and ratios optimal for IBA production in E. coli provide a useful starting point for optimizing production of IBA in diverse microbes and fermentation conditions.

Corrigendum: A Markerless Method for Genome Engineering in Zymomonas mobilis ZM4.

[This corrects the article DOI: 10.3389/fmicb.2019.02216.].

A Markerless Method for Genome Engineering in Zymomonas mobilis ZM4.

Metabolic engineering of the biofuel-producing Zymomonas mobilis is necessary if we are to unlock the metabolic potential present in this non-model microbe. Manipulation of such organisms can be challenging because of the limited genetic tools for iterative genome modification. Here, we have developed an efficient method for generating markerless genomic deletions or additions in Z. mobilis. This is a two-step process that involves homologous recombination of an engineered suicide plasmid bearing Z. mobilis targeting sequences and a subsequent recombination event that leads to loss of the suicide plasmid and a genome modification. A key feature of this strategy is that GFP expressed from the suicide plasmid allows easy identification of cells that have lost the plasmid by using a fluorescence activated cell sorter. Using this method, we demonstrated deletion of the gene encoding lactate dehydrogenase (ldh) and the operon for cellulose synthase (bcsABC). In addition, by modifying the plasmid design, we demonstrated targeted insertion of the crtIBE operon encoding a neurosporene biosynthetic pathway into the Z. mobilis genome without addition of any antibiotic resistance genes. We propose this approach will provide an efficient and flexible platform for improved genetic engineering of Z. mobilis.