RNA polymerase II-driven CRISPR-Cas9 system for efficient non-growth-biased metabolic engineering of Kluyveromyces marxianus.

The thermotolerant yeast Kluyveromyces marxianus has gained significant attention in recent years as a promising microbial candidate for industrial biomanufacturing. Despite several contributions to the expanding molecular toolbox for gene expression and metabolic engineering of K. marxianus, there remains a need for a more efficient and versatile genome editing platform. To address this, we developed a CRISPR-based editing system that enables high efficiency marker-less gene disruptions and integrations using only 40 bp homology arms in NHEJ functional and non-functional K. marxianus strains. The use of a strong RNA polymerase II promoter allows efficient expression of gRNAs flanked by the self-cleaving RNA structures, tRNA and HDV ribozyme, from a single plasmid co-expressing a codon optimized Cas9. Implementing this system resulted in nearly 100% efficiency of gene disruptions in both NHEJ-functional and NHEJ-deficient K. marxianus strains, with donor integration efficiencies reaching 50% and 100% in the two strains, respectively. The high gRNA targeting performance also proved instrumental for selection of engineered strains with lower growth rate but improved polyketide biosynthesis by avoiding an extended outgrowth period, a common method used to enrich for edited cells but that fails to recover advantageous mutants with even slightly impaired fitness. Finally, we provide the first demonstration of simultaneous, markerless integrations at multiple loci in K. marxianus using a 2.6 kb and a 7.6 kb donor, achieving a dual integration efficiency of 25.5% in a NHEJ-deficient strain. These results highlight both the ease of use and general robustness of this system for rapid and flexible metabolic engineering in this non-conventional yeast.

Metabolic engineering of Escherichia coli for optimized biosynthesis of nicotinamide mononucleotide, a noncanonical redox cofactor.

BACKGROUND: Noncanonical redox cofactors are emerging as important tools in cell-free biosynthesis to increase the economic viability, to enable exquisite control, and to expand the range of chemistries accessible. However, these noncanonical redox cofactors need to be biologically synthesized to achieve full integration with renewable biomanufacturing processes. RESULTS: In this work, we engineered Escherichia coli cells to biosynthesize the noncanonical cofactor nicotinamide mononucleotide (NMN+), which has been efficiently used in cell-free biosynthesis. First, we developed a growth-based screening platform to identify effective NMN+ biosynthetic pathways in E. coli. Second, we explored various pathway combinations and host gene disruption to achieve an intracellular level of ~ 1.5 mM NMN+, a 130-fold increase over the cell's basal level, in the best strain, which features a previously uncharacterized nicotinamide phosphoribosyltransferase (NadV) from Ralstonia solanacearum. Last, we revealed mechanisms through which NMN+ accumulation impacts E. coli cell fitness, which sheds light on future work aiming to improve the production of this noncanonical redox cofactor. CONCLUSION: These results further the understanding of effective production and integration of NMN+ into E. coli. This may enable the implementation of NMN+-directed biocatalysis without the need for exogenous cofactor supply.

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

Kluyveromyces marxianus is 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. Unlike Saccharomyces cerevisiae, K. marxianus grows 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 evaluated K. marxianus for 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.